Abstract Background: Xenon, the inert anesthetic gas, is neuroprotective in models of brain injury. The authors investigate the neuroprotective mechanisms of the inert gases such as xenon, argon, krypton, neon, and helium in an in vitro model of traumatic brain injury. Methods: The authors use an in vitro model using mouse organotypic hippocampal brain slices, subjected to a focal mechanical trauma, with injury quantified by propidium iodide fluorescence. Patch clamp electrophysiology is used to investigate the effect of the inert gases on N-methyl-d-aspartate receptors and TREK-1 channels, two molecular targets likely to play a role in neuroprotection. Results: Xenon (50%) and, to a lesser extent, argon (50%) are neuroprotective against traumatic injury when applied after injury (xenon 43 ± 1% protection at 72 h after injury [N = 104]; argon 30 ± 6% protection [N = 44]; mean ± SEM). Helium, neon, and krypton are devoid of neuroprotective effect. Xenon (50%) prevents development of secondary injury up to 48 h after trauma. Argon (50%) attenuates secondary injury, but is less effective than xenon (xenon 50 ± 5% reduction in secondary injury at 72 h after injury [N = 104]; argon 34 ± 8% reduction [N = 44]; mean ± SEM). Glycine reverses the neuroprotective effect of xenon, but not argon, consistent with competitive inhibition at the N-methyl-d-aspartate receptor glycine site mediating xenon neuroprotection against traumatic brain injury. Xenon inhibits N-methyl-d-aspartate receptors and activates TREK-1 channels, whereas argon, krypton, neon, and helium have no effect on these ion channels. Conclusions: Xenon neuroprotection against traumatic brain injury can be reversed by increasing the glycine concentration, consistent with inhibition at the N-methyl-d-aspartate receptor glycine site playing a significant role in xenon neuroprotection. Argon and xenon do not act via the same mechanism.